Next Article in Journal
Multiplex LC-MS/MS Assays for Clinical Bioanalysis of MEDI4276, an Antibody-Drug Conjugate of Tubulysin Analogue Attached via Cleavable Linker to a Biparatopic Humanized Antibody against HER-2
Next Article in Special Issue
Targeted Nanobody-Based Molecular Tracers for Nuclear Imaging and Image-Guided Surgery
Previous Article in Journal
Impact of Acetylated and Non-Acetylated Fucose Analogues on IgG Glycosylation
Previous Article in Special Issue
Selection and Characterization of a Nanobody Biosensor of GTP-Bound RHO Activities
Article Menu

Export Article

Open AccessArticle

A Strategy to Optimize the Generation of Stable Chromobody Cell Lines for Visualization and Quantification of Endogenous Proteins in Living Cells

Pharmaceutical Biotechnology, Eberhard Karls University, 72076 Tuebingen, Germany
Natural and Medical Sciences Institute at the University of Tuebingen, Markwiesenstr. 55, 72770 Reutlingen, Germany
Author to whom correspondence should be addressed.
Antibodies 2019, 8(1), 10;
Received: 4 December 2018 / Revised: 4 January 2019 / Accepted: 7 January 2019 / Published: 10 January 2019
(This article belongs to the Special Issue Nanobody)
PDF [2570 KB, uploaded 10 January 2019]


Single-domain antibodies have emerged as highly versatile nanoprobes for advanced cellular imaging. For real-time visualization of endogenous antigens, fluorescently labelled nanobodies (chromobodies, CBs) are introduced as DNA-encoded expression constructs in living cells. Commonly, CB expression is driven from strong, constitutively active promoters. However, high expression levels are sometimes accompanied by misfolding and aggregation of those intracellular nanoprobes. Moreover, stable cell lines derived from random genomic insertion of CB-encoding transgenes bear the risk of disturbed cellular processes and inhomogeneous CB signal intensities due to gene positioning effects and epigenetic silencing. In this study we propose a strategy to generate optimized CB expressing cell lines. We demonstrate that expression as ubiquitin fusion increases the fraction of intracellularly functional CBs and identified the elongation factor 1α (EF1-α) promoter as highly suited for constitutive CB expression upon long-term cell line cultivation. Finally, we applied a CRISPR/Cas9-based gene editing approach for targeted insertion of CB expression constructs into the adeno-associated virus integration site 1 (AAVS1) safe harbour locus of human cells. Our results indicate that this combinatorial approach facilitates the generation of fully functional and stable CB cell lines for quantitative live-cell imaging of endogenous antigens. View Full-Text
Keywords: nanobodies; chromobodies; live-cell imaging; compound screening; cellular models nanobodies; chromobodies; live-cell imaging; compound screening; cellular models

Figure 1

This is an open access article distributed under the Creative Commons Attribution License which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited (CC BY 4.0).

Supplementary material


Share & Cite This Article

MDPI and ACS Style

Keller, B.-M.; Maier, J.; Weldle, M.; Segan, S.; Traenkle, B.; Rothbauer, U. A Strategy to Optimize the Generation of Stable Chromobody Cell Lines for Visualization and Quantification of Endogenous Proteins in Living Cells. Antibodies 2019, 8, 10.

Show more citation formats Show less citations formats

Note that from the first issue of 2016, MDPI journals use article numbers instead of page numbers. See further details here.

Related Articles

Article Metrics

Article Access Statistics



[Return to top]
Antibodies EISSN 2073-4468 Published by MDPI AG, Basel, Switzerland RSS E-Mail Table of Contents Alert
Back to Top